Most cultivation methods for the growth of microbial prokaryotic or eukaryotic cells are based on the cultivation in a liquid medium. In practice, such liquid cultures are performed in a batch process operational mode. The batch process is a discontinuous process, where the sterile growth medium with all required substrates is initially inoculated with a pure culture of microbial prokaryotic or eukaryotic cells and no additional growth medium is added during the course of operation. This means, that the batch process is a partially closed system, wherein the only material added and removed during the course of operation is air/gas exchange, antifoam and pH controlling agents (Cinar A. et al., Batch fermentation—modeling, monitoring, and control, 2003, Marcel Dekker Inc., page 5). These batch cultures are continuously shaken or stirred to keep a desired degree of homogeneity of the substrates and cells to guarantee as high as possible oxygen transfer for aerobic cultures. These non-controlled shaken batch cultures, however, have substantial disadvantages, e.g. the high initial substrate concentrations in the growth medium. This high initial substrate concentration leads to long adaptation phases (lag phases) of the microbial prokaryotic or eukaryotic cells, which are especially relevant in enrichment cultures, e.g. in food diagnostics. In high substrate concentrations the microbial prokaryotic or eukaryotic cells may respond with overflow metabolism and secretion of large amounts of by-products, mainly acetate, ethanol, and lactate. Non-controlled growth also easily leads to oxygen deprivation and if anaerobic conditions occur the microbial prokaryotic or eukaryotic cells also secrete formate, succinate, hydrogen, and additional CO2 (Luli W. R. & W. R. Strohl, Appl. Environ. Microbiol., 1990, 56:1004-1011; Riesenberg D. et al., J. Biotechnol., 1991, 20:17-28). Thus, anaerobic metabolism (fermentation reactions) and overflow metabolism cause a drift of pH and secretion of fermentation products in amounts, which may inhibit the growth of microbial prokaryotic or eukaryotic cells and impair recombinant protein production. Some of these metabolites are also synthesized under aerobic conditions if increasing concentrations of substrates, e.g. carbohydrates, repress the genes of respiration. Thus, cells which normally grow fast with oxygen as terminal electron acceptor, will show growth inhibition and side metabolite accumulation (ethanol), even under aerobic conditions (glycolysis) when the substrate concentration is high, a phenomenon called “Crabtree effect” (Crabtree H. G., J. Biochem., 1928, 22:1289-1298; Rinas U. et al., Appl. Microbiol. Biotechnol., 1989, 31:163-167). Long-term exposure to high substrate concentrations is characterized by catabolite repression; the substrates that provide the cells with the most energy and growth advantage are selectively taken up, whereas various functions involved in the catabolism and uptake of the less preferred substrates are repressed (Monod J., Actualites scientifiques et industrielles, 1942, 911:70-78). This leads to low biomass yield, and poor quality and quantity of microbial prokaryotic or eukaryotic cell products. The biomass yield in shaken Escherichia coli cultures is typically only in the range of 1-2 g dry cells per liter in shake flask cultures, and in microscale often much lower. Thus, high cell densities are not achieved with the batch technology.
In order to avoid the above mentioned effects of high initial substrate concentration, most large-scale cultivations in bioreactors apply the fed-batch technology (Kleman G. L. & Strohl W. R., Curr. Opin. Biotechnol., 1994, 5:180-186; Riesenberg D., Curr. Opin. Biotechnol., 1991, 2:380-384). Fed-batch cultivation is distinguished from batch cultivation by the addition of a defined amount of fresh growth-limiting substrate in highly concentrated form, mostly by continuous feeding (Kleman G. L. & Strohl W. R., Curr. Opin. Biotechnol., 1994, 5:180-186). In industrial practice the process efficiency can be increased by regular withdrawal of the cultivation broth, a procedure which is called repeated fed-batch (Longbardi G. P., Bioproc. Engin., 1994, 10:185-194). Oxygen limitation, pH drift and the inhibition of growth due to fermentation by-products can be avoided with continuous substrate feeding because the oxygen consumption increases relative to the substrate consumption rate and the growth rate of the culture. With well-controlled substrate feeding, high cell densities with up to 50-fold higher biomass compared to batch cultivations can be produced in commonly used industrial bioreactors. E. coli cultures can reach final biomass concentrations of more than 100 g dry cells per liter (Lee S. Y., Trends Biotechnol., 1996, 14:98-105; Riesenberg D., Curr. Opin. Biotechnol., 1991, 2:380-384). Although the fed-batch technology is very well applicable in industrial bioreactors, it is not easily applicable for small laboratory-scale shaken cultures. Thus, alternative fed-batch cultivation strategies have been developed. In the following a difference is made between 1) growth-limiting substrate-monomers, which are metabolically active, 2) substrate-oligomers, and 3) substrate-polymers, which are metabolically inactive.
For example, in the field of medical technology drugs are often supplied by the fed-batch slow-release technique, also called delivery systems. These systems are based on the slow release of a metabolically active growth-limiting substrate-monomer by diffusion from a solid phase, e.g. from artificial polymer matrices, over a long period of time. Such polymer matrices can also be packed with nutrient components and they can be added to cultivation vessels. With such a fed-batch slow-release technique, Lübbe C. et al. (Appl. Microbiol. Biotechnol., 1985, 22:424-427) fed ammonia to Streptomyces clavuligerus cultivation from ethylene-vinylacetate copolymer discs containing NH4Cl to study NH4+ control and to increase the production of cephalosporins in comparison to batch cultures. However, the authors did not observe significant advantage of applying their fed-batch slow-release system because they were not able to match the exponential growth of the cells with a fixed, linear feed of the growth-limiting substrate-monomer. Furthermore, Jeude M. et al. (Biotechnol. Bioeng., 2006, 95:433-445) used silicone elastomer (polydimethylsiloxane) discs as a solid phase containing the growth-limiting substrate-monomer to create fed-batch like conditions for cultivations (see also Büchs J. et al., WO2006/119867). Although the authors observed minimization of overflow metabolism, which resulted in a higher biomass yield, these systems are rarely applied in microbial prokaryotic or eukaryotic cell cultivations. This is because only relatively small amounts of the growth-limiting substrate-monomer can be packed into such solid phases. Furthermore, the substrate-monomer release rate from such a solid phase is usually fastest at the beginning of the cultivation, when the amount of microbial prokaryotic or eukaryotic cells is lowest and the risk for overflow metabolism is highest. Thus, the fed-batch slow-release cultivation approaches based on solid phases that directly release the growth-limiting substrate-monomer into the medium are limited with regards to the scalability, i.e. to the amount of the growth-limiting substrate-monomer that can be packed to the system and the possibilities to accurately control the substrate-monomer release. Moreover, such solid phases are not easy to produce, which limit their applicability.
The enzyme-based fed-batch system of Vasala A. et al. (PCT/FI2007/050648) offers a much better capacity and an excellent control of substrate release and microbial prokaryotic or eukaryotic cell growth (see also Panula-Perälä J. et al., 2007, J. Biotechnol., 131S:S182—Issue for the 13th European Congress of Biotechnology, Barcelona, Spain, poster no. 91.-doi:10.1016/j.jbiotec.2007.07.920, Panula-Perälä et al., 2008, Microb. Cell Fact. 7:31). There, a fed-batch technology is described, having a liquid phase and a solid phase, i.e. a two-phase system. In difference to the previous approaches the solid phase, e.g. a gel phase, provides a source of a metabolically inactive substrate-polymer, which delivers the metabolically active substrate-monomer by biocatalytic degradation, i.e. enzyme-based. In this system the delivery rate of the growth substrate and thus the growth rate of the microbial prokaryotic or eukaryotic cells can be simply controlled by the concentration of the substrate-polymer degrading enzyme. Such a system has advantages in comparison to usual fed-batch slow-release systems comprising a solid phase because the release of the growth-limiting substrate-monomer to the liquid phase is retarded and can be simply controlled. With this method a high amount of substrate-polymer can be packed into the system. Furthermore, the gel-formulation as the solid phase ensured that most of the substrate-polymer is maintained in a water-soluble form. Thus, high cell densities are supported without impairing the physical properties of the liquid phase. However, this system has also disadvantages insofar as starch immobilized into a gel is slowly diffusing into a liquid phase, and simultaneously being degraded enzymatically. In addition, the capacity of the gel seriously limits the amount of starch that can be applied to the system. Due to the presence of a gel, not only the enzyme amount but also the starch diffusion rate determines the reaction speed. Too fast starch diffusion may result in accumulation of insoluble starch into the liquid phase which has negative effects on the cell growth.
All the fed-batch systems described above are composed of a two-phase system providing a liquid phase, which contains the microbial prokaryotic or eukaryotic cells and the cultivation medium, and a solid phase, which contains the growth-limiting substrate-monomer or the substrate-polymer. However, two-phase systems are not easy to use in biotechnological applications where frequent (automatic) samplings or measurements are needed. This means that two-phase systems are not only complicated to produce but also limited in their applicability in many biotechnological applications, e.g. laboratory-scale. This may explain why such systems have not become popular in simple cultivations of the biotechnologically most important bacterial species, E. coli. 
For microbial prokaryotic or eukaryotic cells, which cannot efficiently degrade substrate-polymers in the medium, a method of partial or complete enzymatic degradation of substrate-polymers has been developed to enhance their growth rate. For example, Tokuda M. et al. (J. Ferment. Bioeng., 1998, 85:495-501) showed that anaerobic methane fermentation of whiskey distillery waste can be enhanced by partial digestion of starch (substrate-polymer) with enzymes or with moulds prior to anaerobic methane production process. This kind of enzymatic pretreatment, however, is not suitable to provide a controlled cultivation for obtaining high cell densities of microbial prokaryotic or eukaryotic cells.
Another interesting application for the cultivation of eukaryotic cells has been presented by Green H. & J. G. Rheinwald (U.S. Pat. No. 3,926,723). The aim of the authors was to improve the cell yield in mammalian cell cultures by decreasing the accumulation of harmful metabolites. The authors reasoned that low concentrations of the growth-limiting substrate-monomer (glucose) cannot be maintained by direct addition to the medium because the growing cells consume them rapidly. Therefore, they used small amounts of a substrate-polymer (starch) in a liquid medium and activities of hydrolytic enzymes (e.g. amylase and maltase) present in horse, pig, or bovine serum to release growth-limiting monomers (glucose), i.e. cultivations were done in rich medium containing serum. This is no chemically defined medium and makes it impossible to control the cell growth. The authors used only 1 g/l of starch, which in theory would support at most 1 g dry cells per liter of biomass (cells). In practice, cell yields remained considerably lower because starch easily looses its solubility and digestibility in water-based liquids. As a result, no significant increase of cell number was obtained. Thus, one can conclude that such technique was not used for controlling or enhancing the growth rate of eukaryotic cells but to prevent the accumulation of harmful metabolites (e.g. lactic acid) as a growth-retarding compound. Therefore, it cannot be regarded as a method for enzyme-based fed-batch high-cell-density cultivation.
Another approach where enzymatic degradation of cellulosic material was used to provide a carbon source for microorganisms has been described by Asenjo et al. (Asenjo et al., Biotechnology and Bioengineering, Vol. 37 (1991), pp. 1087-1094; Asenjo et al., Bioprocess Engineering 14 (1996), pp. 323-329) Their research was run to optimize product formation by minimizing the accumulation and inhibitory effect of intermediate compounds (glucose or cellubiose). Asenjo et al. optimized enzyme feeding so that enzyme-inhibiting compounds will not accumulate. This approach, however, does not lead to high cell density cultivation
The object of the present invention is to provide a method for continuous and high-cell-density microbial prokaryotic or eukaryotic cell cultivation in laboratory- or large-scale liquid shaken cultures having the possibility to control the growth rate of the cultured organisms by a controlled enzymatic release of the growth-limiting substrate-monomer from substrate-polymers or substrate-oligomers.